SAT-166 Techniques For Direct Imaging Of Beta-cell Activity And Glucose Homeostasis In Zebrafish

Zebrafish have recently gained popularity as model organisms for the study of insulin secretory control and diabetes, and their beta-cells (b-cells) have remarkable genetic and physiologic conservation to mammals [1]. Zebrafish have significant advantages over mammalian models, due to their translucent embryos and large number of progeny, allowing for large-scale genetic and pharmacologic screens. However, monitoring glucose levels in fish requires either time-consuming blood draws or sacrificing of the animal, and attempts by multiple groups at directly measuring insulin secretion have been unsuccessful. To optimize zebrafish as a model organism, we are developing approaches for less invasive determinations of glucose levels and for quantitative imaging of glucose stimulated b-cell activity. Insulin secretion is regulated by ATP-sensitive potassium (KATP) channels. At low plasma [glucose], KATP channels are open and the membrane is hyperpolarized, thus inhibiting insulin secretion. Increased glucose metabolism raises the intracellular ATP, closing KATP channels. This leads to membrane depolarization, calcium influx, and ultimately triggers insulin release. Genetic disruption of this pathway can lead to neonatal diabetes or congenital hyperinsulinism. We have generated b-cell specific GCaMP transgenic fish to allow detection of intracellular [Ca2+]. We demonstrate a 2-fold increase in intracellular [Ca2+] when switching from 2mM glucose to 20mM. This increase is fully inhibited when incubated in 20 mM glucose with 250 uM diazoxide. To assess secretion, islets were labeled with a fluorescent zinc indicator [2] and exposed to increasing [glucose]. Fluorescence increases 10-25% over baseline in response to high glucose concentration, indicating secretion of zinc-containing insulin vesicles. Increase in fluorescence is fully inhibited with 250 uM diazoxide. We are also evaluating a genetically encoded glucose indicator, created by our collaborators through modification of a bacterial glucose binding protein and GFP. When expressed in Cos cells, a cytosolic form shows a 2-fold fluorescence increase in the range of 10-30 mM glucose. We have generated transgenic lines for this biosensor allowing detection of real time, in vivo changes in [glucose] without sacrificing the animal. Establishment of these techniques will facilitate the use of zebrafish as a model organism for the study of disorders of insulin secretion and identify novel treatment approaches. 1. Emfinger, C.H., et al., Expression and function of ATP-dependent potassium channels in zebrafish islet beta-cells. R Soc Open Sci, 2017. 4(2): p. 160808. 2. Li, D., et al., Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR). Proc Natl Acad Sci U S A, 2011. 108(52): p. 21063-8.